lecture 4: reaction mechanisms and inhibitors reaction mechanisms a: sequential reactions all...

LECTURE 4:Reaction

Mechanisms and Inhibitors

Reaction MechanismsA: Sequential Reactions• All substrates must combine with

enzyme before reaction can occur

Bisubstrate reactions

B. Random Bisubstrate Reactions

C. Ping-Pong Reactions• Group transfer reactions• One or more products released

before all substrates added

Kinetic data cannot unambiguously establish a reaction mechanism.

Although a phenomenological description can be obtained the nature of the reaction intermediates remain indeterminate and other independent measurements are needed.

QUIZ (10 min)1. How is enzyme specificity achieved ?2. Calculate Vmax & KM from the following data, and

does the reaction obey Michaelis-Menten kinetics ?

[DNA]mol total

nucleotides/L

Free nucleotides in solution,V (pmol/L)

0 min 10 min1.0 x 10-5 0.05 5.11.0 x 10-6 0.04 4.51.0 x 10-7 0.06 3.21.0 x 10-8 0.04 1.41.0 x 10-9 0.04 0.23

ANSWERS1. The enzyme specificity is

achieved through the characteristic of active site

2. Vmax = 4.36695 KM = 2.2E-08 R2 = 0.999864, so the

reaction obeys Michaelis-Menten kinetics

• An important number of compounds have the ability to combine with certain enzymes in either a reversible or irreversible manner, and thereby block catalysis by that enzyme

• Such compounds are called INHIBITORS and include drugs, antibiotics, poisons, anti metabolites, as well as products of enzymic reactions

• Two general classes of inhibitors are recognized ; – Irreversible – Reversible

INHIBITORS

• An irreversible inhibitor forms a covalent bond with a specific function, usually an amino acid residue, which may, in some manner, be associated with the catalytic activity of the enzyme

• There are many examples of enzyme inhibitors which covalently bind not at the active site, but physically block the active site

• The inhibitor cannot be released by dilution or dialysis; kinetically, the concentration and hence the velocity of active enzyme is lowered in proportion to the concentration of the inhibitor and thus the effect is that of noncompetitive inhibition:

1. IRREVERSIBLE INHIBITORS

Irreversible Inhibition E + S ES E + P + I EI

KS

KI

• Examples of irreversible inhibitors include diisopropyl fluorophosphate, which reacts irreversibly with serine proteases, chymotrypsin and iodoacetate which reacts with essential sulfhydryl group of an enzyme such as triose phosphate dehydrogenase:

E-SH+ICH2COOH E-SCH2COOH+HI• A unique type of irreversible inhibition

has been recently described as kcat inhibition in that a latent inhibitor is activated to an active inhibitor by binding to the active site of the enzyme.

• The newly generated inhibitor now reacts chemically with the enzyme leading to its irreversible inhibition

• These inhibitors have great potential as drugs in highly specific probes for active sites since they are not converted from the latent to the active form except by their specific target enzymes

• An excellent example is the inhibition of D‑3‑hydroxyl decanoyl ACP clehydrase (of E. coli) by the latent inhibitor 3‑decynoyl‑N‑acetyl cystamine according to the following sequences of events:

2. REVERSIBLE INHIBITION• As the term implies, this type of inhibition

involves equilibrium between the enzyme and the inhibitor, the equilibrium constant (Ki) being a measure of the affinity of the inhibitor for the enzyme.

• Three distinct types of reversible inhibition are known; – Competitive inhibition,– Noncompetitive inhibition – Uncompetitive inhibition.

A. Competitive Inhibition • Compounds that may or may not be

structurally related to the natural substrate combine reversibly with the enzyme at or near the active site

• The inhibitor and the substrate therefore compete for the same site according to the reaction:

]S[K

]I[1K

]S[VV

IM

max

Competitive Inhibition E + S ES E + P + I EI

KS

KI

ES and EI complexes are formed, but EIS complexes are never produced. One can conclude that high concentrations of substrate will overcome the inhibition by causing the reaction sequence to swing to the right. The velocity of reaction can be calculated by the following equation

C o m p etitiv e in h ib ito r

-I

+I

-1/KM -1/[KM(1+1/KI)]

1/V

1/S

• Among other enzymes that may undergo competitive inhibition (Table 1) is succinic dehydrogenase, which readily oxidizes succinic acid to fumaric acid.

• If increasing concentrations of malonic acid, which closely resembles succinic acid in structure, are added, however, succinic dehydrogenase activity falls markedly. This inhibition can now be reversed by increasing in turn the concentration of the substrate succinic acid.

B. Noncompetitive Inhibition • Compounds that reversibly bind with either the

enzyme or the enzyme substrate complex are designated as noncompetitive inhibitors and the following reactions describe these events:

Noncompetitive Inhibition E + S ES E + P + + I I EI + S EIS

KS

KI KI KS

• Noncompetitive inhibition therefore differs from competitive inhibition in that the inhibitor can combine with ES, and S can combine with EI to form in both instances EIS.

• This type of inhibition is not completely reversed by high substrate concentration since the closed sequence will occur regardless of the substrate concentration.

• Since the inhibitor binding site is not identical to nor does it modify the active site directly, the KM is not altered. The equation used to calculate the velocity of the noncompetitive inhibition is as follows

IM

max

K]I[1]S[K

]S[VV

N oncom petitive

-I

+ I

-1 /V m ax

(1+ [I]/K I)/V m ax

1/V

1/S

C. Uncompetitive Inhibition • Compounds that combine only with the ES

complex but not with the free enzyme are called uncompetitive inhibitors. The inhibition is not overcome by high substrate concentrations.

Uncompetitive Inhibition E + S ES E + P + I EIS

KS

KI

• Interestingly the KM value is consistently smaller than the KM value of the uninhibited reaction, which implies that S is more effectively bound to the enzyme in the presence of the inhibitor.

• The equation used to calculate the velocity of the noncompetitive inhibition is as follows

IM

max

K]I[1]S[K

]S[VV

Uncompetitive inhibitor

-I

+I

-1/Vmax

(1+[I]/KI)/Vmax

-(1+[I]/KI)/KM

-1/KM

1/S

1/V

FEEDBACK INHIBITION

HOW TO SOLVE THE EQUATIONS

1. Competitive inhibitor

•y =1/V; x = 1/[s]•a = 1/Vmax•b = KM(1+[I]/KI)/Vmax

]S[K

]I[1K

]S[VV

IM

max

maxV1

]S[1

VK

]I[1K

V1

max

IM

2. Noncompetitive Inhibition

•y =1/V; x = 1/[s]•a = (1+[I]/KI)/Vmax•b = KM(1+[I]/KI)/Vmax

IM

max

K]I[1]S[K

]S[VV

maxVK

]I[1

]S[1

maxVK

]I[1K

V1 II

M

3. Uncompetitive

•y =1/V; x = 1/[s]•a = (1+[I]/KI)/Vmax•b = KM/Vmax

IM

max

K]I[1]S[K

]S[VV

maxVK

]I[1

]S[1

maxVK

V1 IM

SOAL

• Diketahui suatu reaksi enzimatis tanpa dan dengan inhibitor dengan [I] = 2,2.104M.

• Hitunglah KM dan Vmax tanpa dan dengan I serta KI

[S] V(-I) V(+I)

1*10-4 28 17

1.5*10-4 36 23

2.0*10-4 43 29

5*10-4 65 50

7.5*10-4 74 61

THANK YOU